JP4915722B2 - Separation method of kidney stem / progenitor cells - Google Patents

Separation method of kidney stem / progenitor cells Download PDF

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JP4915722B2
JP4915722B2 JP2006027360A JP2006027360A JP4915722B2 JP 4915722 B2 JP4915722 B2 JP 4915722B2 JP 2006027360 A JP2006027360 A JP 2006027360A JP 2006027360 A JP2006027360 A JP 2006027360A JP 4915722 B2 JP4915722 B2 JP 4915722B2
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健 菅谷
尚史 篠崎
伸一 大島
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株式会社 バイオリンクインク
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本発明は、ヒトの腎臓幹／前駆細胞の分離方法に関する。 The present invention relates to the separation how human renal stem / progenitor cells.

近年、慢性腎不全の患者数が増加している。慢性腎不全は、腎臓における疾病又は腎臓以外の器官における疾病が原因で生じた腎臓障害が進行した病態であり、血液中に含有される尿毒素を除去する機能、内分泌機能、調節機能など、正常な腎臓が有する機能が廃絶する。   In recent years, the number of patients with chronic renal failure has increased. Chronic renal failure is a condition in which kidney damage caused by a disease in the kidney or a disease in an organ other than the kidney has progressed and is normal, such as a function to remove uremic toxins contained in blood, an endocrine function, and a regulatory function The function of a healthy kidney is abolished.

慢性腎不全が進行すると、正常な腎臓が有する機能が廃絶するために、体内の各臓器に影響が及び、***となる。具体的には、中枢神経障害、末梢神経障害、心・循環器障害、消化器障害、視力・眼障害、血液・凝固障害、免疫障害、内分泌障害、皮膚障害、骨・関節障害、電解質障害、酸塩基平衡障害などになる。   As chronic renal failure progresses, the functions of a normal kidney are abolished, affecting each organ in the body and causing uremia. Specifically, CNS disorders, peripheral nerve disorders, cardiovascular disorders, digestive disorders, vision / eye disorders, blood / coagulopathy, immune disorders, endocrine disorders, skin disorders, bone / joint disorders, electrolyte disorders, It becomes an acid-base balance disorder.

慢性腎不全は、腎臓移植が現存する唯一の根治療法であるが、ドナー不足のため全ての慢性腎不全患者に移植を施すことは不可能である。このため、慢性腎不全患者は生存のためには人工透析を余儀なくされ、この透析患者の増大が医療費の増大を招き、大きな社会問題にもなっている。また、人工透析は、技術の進歩によって患者への負担は以前よりも軽減されてきているものの、腎臓移植を受けて根治した患者と比べると、その生活の質（ＱＯＬ）においては格段の差がある。   Chronic renal failure is the only radical cure that currently exists, but it is not possible to transplant all chronic renal failure patients due to a lack of donors. For this reason, chronic renal failure patients are forced to undergo artificial dialysis in order to survive, and the increase in the number of dialysis patients has led to an increase in medical costs and has become a major social problem. Although artificial dialysis has reduced the burden on patients as a result of technological advances, there is a marked difference in quality of life (QOL) compared to patients who have undergone kidney transplantation and have been cured. is there.

Poulsom R. et al.,“Bone marrow cells contribute to both normal turnover of renal epithelia and regeneration after damage.”, J. Pathol., 195, pp.229-235, 2001Poulsom R. et al., “Bone marrow cells contribute to both normal turnover of renal epithelia and regeneration after damage.”, J. Pathol., 195, pp.229-235, 2001 Bussolati B. et al.,“CD133+ cells from kidney represent a multipotent adult resident stem cell population that may contribute to the repair of renal injury.”, Am. J. Pathol., 166, pp.545-555, 2005Bussolati B. et al., “CD133 + cells from kidney represent a multipotent adult resident stem cell population that may contribute to the repair of renal injury.”, Am. J. Pathol., 166, pp.545-555, 2005

ところで、近年、幹／前駆細胞を用いて組織や臓器をin vitro又はin vivoで形成することで病変や欠損を治療する、いわゆる再生医療が注目を集めている。このような流れの中で、骨髄中や腎臓に腎臓幹／前駆細胞が存在することが明らかにされている（非特許文献１，２を参照）。   By the way, in recent years, so-called regenerative medicine, which treats lesions and defects by forming tissues and organs in vitro or in vivo using stem / progenitor cells, has attracted attention. In such a flow, it has been clarified that kidney stem / progenitor cells exist in the bone marrow and in the kidney (see Non-Patent Documents 1 and 2).

そこで、この腎臓幹／前駆細胞を分離・取得して再生医療に応用することが考えられるが、骨髄中や腎臓から腎臓幹／前駆細胞を分離するのは、生体に対する負担が大きく、すなわち侵襲性が高いという問題があった。   Therefore, it is conceivable to isolate and acquire these kidney stem / progenitor cells and apply them to regenerative medicine. However, separating kidney stem / progenitor cells from bone marrow and kidneys is a heavy burden on the living body, that is, is invasive. There was a problem of high.

本発明は、このような従来の実情に鑑みて提案されたものであり、ヒトの腎臓幹／前駆細胞を非侵襲的に分離する方法を提供することを目的とする。 The present invention has such a proposed in view of the conventional circumstances, and an object thereof is to provide a way to separate the human renal stem / progenitor cells non-invasively.

本件発明者等は、上述した目的を達成するために、様々な観点から鋭意研究を重ねてきた。その結果、腎機能が低下している腎疾患患者の尿中に落下している尿中落下細胞から腎臓幹／前駆細胞を分離・取得できることを見出した。本発明は、このような知見に基づいて完成されたものである。   In order to achieve the above-described object, the present inventors have conducted intensive research from various viewpoints. As a result, the present inventors have found that kidney stem / progenitor cells can be separated and obtained from urine falling cells falling in the urine of kidney disease patients whose renal function is reduced. The present invention has been completed based on such findings.

すなわち、本発明に係る腎臓幹／前駆細胞の分離方法は、腎疾患患者の尿中に含まれる細胞の初代培養細胞をヘキスト３３３４２で染色したときの弱陽性又は陰性画分を分離することを特徴とする。   That is, the method for separating kidney stem / progenitor cells according to the present invention is characterized by separating a weak positive or negative fraction when primary cultured cells of cells contained in the urine of a renal disease patient are stained with Hoechst 33342. And

従来、腎疾患の指標としては、血中クレアチニン、シスタチンＣ、ＢＵＮ（blood urea nitrogen；血中尿素窒素）などが知られているが、近年、尿や血液に含まれる脂肪酸結合タンパク質（fatty acid binding protein；ＦＡＢＰ）のうち、肝型の分子種（以下、Ｌ−ＦＡＢＰという。）が、腎臓では近位尿細管に特異的に発現しており、腎疾患の指標となり得ることが報告されている（特許第３２５９７５８号公報を参照）。すなわち、尿中Ｌ−ＦＡＢＰ値の正常上限は１０ｎｇ／ｍｌ程度であるが、腎疾患患者では例えば５０ｎｇ／ｍｌ以上など、高値を示すため、腎疾患の指標として用いることができる。特に、尿中Ｌ−ＦＡＢＰ値とヘキスト３３３４２弱陽性／陰性画分の割合とが正に相関することが本件発明者によって確認されているため、本発明においては、このＬ−ＦＡＢＰを指標として腎疾患患者を選ぶことが好ましい。   Conventionally, blood creatinine, cystatin C, BUN (blood urea nitrogen) and the like are known as indicators of kidney disease, but in recent years, fatty acid binding protein (fatty acid binding protein contained in urine and blood) It has been reported that a liver-type molecular species (hereinafter referred to as L-FABP) among proteins (FABP) is specifically expressed in the proximal tubule in the kidney and can be an indicator of renal disease. (See Japanese Patent No. 3259758). That is, the normal upper limit of the urinary L-FABP value is about 10 ng / ml, but it shows a high value such as 50 ng / ml or more in patients with renal disease, and can therefore be used as an indicator of renal disease. In particular, since the present inventors have confirmed that the urinary L-FABP value and the Hoechst 33342 weakly positive / negative fraction ratio are positively correlated, in the present invention, this L-FABP is used as an index for the kidney. It is preferable to select a patient with a disease.

なお、本発明における腎疾患患者には、生体腎移植手術直後の患者や腎機能が未熟な新生児も含まれるものとする。   In addition, the renal disease patient in this invention shall include the patient immediately after living body renal transplant surgery, and the newborn infant with immature renal function.

このような生体腎移植手術直後の患者や腎機能が未熟な新生児も、尿中のＬ−ＦＡＢＰが高値（例えば、１００ｎｇ／ｍｌ以上）を示すため、この指標を用いて尿中から腎臓幹／前駆細胞を効率的に分離することができる。   Such patients immediately after living-donor kidney transplantation and neonates with immature renal function also show high levels of urinary L-FABP (for example, 100 ng / ml or more). Progenitor cells can be efficiently separated.

また、本発明に係る腎臓幹／前駆細胞は、上述の分離方法によって分離されたことを特徴とし、本発明に係る腎疾患治療剤は、上述の分離方法によって分離された腎臓幹／前駆細胞を含むことを特徴とする。   Further, the kidney stem / progenitor cell according to the present invention is separated by the above-described separation method, and the renal disease therapeutic agent according to the present invention comprises the kidney stem / progenitor cell separated by the above-described separation method. It is characterized by including.

本発明によれば、腎疾患患者から非侵襲的に腎臓幹／前駆細胞を分離することができる。 According to the present invention, it is Rukoto from renal disease patient non-invasively release min renal stem / progenitor cells.

以下、本発明を適用した実施の形態について、具体的な実験結果を参照しながら詳細に説明する。   Hereinafter, embodiments to which the present invention is applied will be described in detail with reference to specific experimental results.

尿中落下細胞の培養
先ず、腎疾患患者の尿中から尿中落下細胞を分離し、培養した。 Cultivation of fallen cells in urine First, fallen cells in urine were separated from urine of kidney disease patients and cultured.

詳細には、生体腎移植手術直後の患者の尿１０ｍｌを４℃、１１００ｒｐｍの条件で５分間遠心分離し、上清を除去した後、１０％ ＦＢＳ（Fetal Bovine Serum）（MBL）を含有するＤＭＥＭ（Dulbecco's Modified Eagle's Medium）（Sigma） １０ｍｌで懸濁し、再度遠心分離して上清を除去して細胞を分離した。さらに、この懸濁、遠心分離、上清除去による細胞洗浄の操作を４回繰り返した。このようにして分離された細胞を、１０％ ＦＢＳを含有するＤＭＥＭ／Ｆ１２（Sigma）に終濃度として５ｍｇ／ｍｌ インスリン、５ｍｇ／ｍｌ トランスフェリン、５ｎｇ／ｍｌ 亜セレン酸ナトリウム、２．５ｍｇ／ｍｌ ニコチナマイド、１０−８Ｍ デキサメタゾン（以上、全てSigma）を添加した培地と、マウス間葉系細胞（独立行政法人産業総合研究所 特許生物寄託センターに寄託、受領番号FERM AP-20769）を１０％ ＦＢＳを含有するＤＭＥＭで約２４時間培養した際の培養上清とを１：１で混合した培地 １０ｍｌで懸濁し、ゼラチンコーティングを行った細胞培養皿（BD FALCON）にて培養した。 Specifically, 10 ml of urine from a patient immediately after living kidney transplantation surgery was centrifuged at 4 ° C. and 1100 rpm for 5 minutes, the supernatant was removed, and then DMEM containing 10% FBS (Fetal Bovine Serum) (MBL) (Dulbecco's Modified Eagle's Medium) (Sigma) Suspended in 10 ml, centrifuged again to remove the supernatant, and the cells were separated. Furthermore, this cell washing operation by suspension, centrifugation, and supernatant removal was repeated four times. The cells thus isolated were added to DMEM / F12 (Sigma) containing 10% FBS as a final concentration of 5 mg / ml insulin, 5 mg / ml transferrin, 5 ng / ml sodium selenite, 2.5 mg / ml nicotinamide. 10-8 M Dexamethasone (all above, all Sigma) added medium and mouse mesenchymal cells (deposited with the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, receipt number FERM AP-20769) with 10% FBS It was suspended in 10 ml of a medium mixed with the culture supernatant obtained by culturing in the contained DMEM for about 24 hours, and cultured in a gelatin-coated cell culture dish (BD FALCON).

細胞を経時的に顕微鏡観察した結果を図１に示す。図１（Ａ）〜（Ｅ）は、それぞれ３日、４日、６日、８日、１５日経過後の細胞を示したものである。細胞は培養開始後２４時間以内に培養皿上に生着し、以後１２〜２４時間毎に細胞***を行い、培養開始から２週間後には約５×１０６個程度の細胞数にまで増殖した。このことから、この培養方法により、増殖能を有する細胞が得られることが確認された。 The results of microscopic observation of the cells over time are shown in FIG. 1A to 1E show the cells after 3 days, 4 days, 6 days, 8 days, and 15 days, respectively. The cells were engrafted on the culture dish within 24 hours after the start of the culture, and thereafter divided every 12 to 24 hours. After 2 weeks from the start of the culture, the cells grew to about 5 × 10 6 cells. . From this, it was confirmed that cells having proliferation ability can be obtained by this culture method.

また、これらの増殖能を有する細胞群の中には、敷石上形態を示す尿細管上皮細胞様の細胞からなるコロニーが観察され、図２に示すように、経上皮輸送能を示唆する細胞ドームの形成も確認された。   In addition, colonies composed of tubular epithelial cell-like cells exhibiting a cobblestone morphology are observed in these cell groups having proliferative ability. As shown in FIG. 2, a cell dome suggesting transepithelial transport ability is observed. The formation of was also confirmed.

これらの結果より、生体腎移植手術直後の患者の尿から分離した尿中落下細胞を培養することにより、尿細管細胞様の細胞を取得、培養可能であることが示された。   From these results, it was shown that tubule cell-like cells can be obtained and cultured by culturing falling cells in urine separated from urine of patients immediately after living kidney transplantation surgery.

尿中落下細胞の遺伝子発現解析
次に、分離した尿中落下細胞における遺伝子発現を確認した。 Next, the gene expression in the urine falling cells was confirmed.

詳細には、上述のようにして初代培養を５週間続けた細胞１．５×１０６個に、ＩＳＯＧＥＮ（ニッポンジーン） １ｍｌを加えて懸濁し、さらに０．２ｍｌのクロロホルムを加えてよく混合した後、４℃、１２０００ｒｐｍの条件で１０分間遠心分離した。そして、上清に０．５ｍｌのイソプロパノールを加えてよく混合してから、４℃、１２０００ｒｐｍの条件で２０分間遠心分離し、上清を除去してから１ｍｌの７５％ エタノールで洗浄した後、風乾させた。このようにして得られたtotal ＲＮＡをサンプルとして、遺伝子チップ（Human Genome U133 Plus 2.0 Array Gene Chip、Affymetrix）を用いて遺伝子発現解析を行った。確認した遺伝子と、その陽性／陰性の別を以下の表１に示す。 Specifically, after adding 1.5 ml of ISOGEN (Nippon Gene) and suspending in 1.5 × 10 6 cells that have been maintained in the primary culture for 5 weeks as described above, and then adding 0.2 ml of chloroform and mixing well. Centrifugation was performed at 4 ° C. and 12000 rpm for 10 minutes. Add 0.5 ml of isopropanol to the supernatant and mix well, then centrifuge at 4 ° C. and 12000 rpm for 20 minutes, remove the supernatant, wash with 1 ml of 75% ethanol, and air dry. I let you. Using the total RNA thus obtained as a sample, gene expression analysis was performed using a gene chip (Human Genome U133 Plus 2.0 Array Gene Chip, Affymetrix). The confirmed genes and their positive / negative distinctions are shown in Table 1 below.

Figure 0004915722
Figure 0004915722

表１から分かるように、尿中落下細胞においては、アクアポリン１、Ｎａ＋／Ｃｌ−共輸送体（ＫＣＣ３ａ）、Ｎ−カドヘリンなど、近位尿細管特異的な遺伝子の発現が確認された。また、近位尿細管及びヘンレループで発現するＮａ＋／ＨＣＯ３ −共輸送体（ＳＬＣ４Ａ４）、Ｋ−カドヘリン、ヘンレループ及び遠位尿細管で発現するＮａ＋／ＨＣＯ３ −共輸送体（ＳＬＣ４Ａ７）、遠位尿細管で特異的に発現するカルビンジンＤ２８Ｋなどの遺伝子発現も確認された。その一方で、ネフリンやＰ−カドヘリンなどの腎小体特異的な遺伝子や、アクアポリン２やＮａ＋／ＨＣＯ３ −共輸送体（ＳＬＣ４Ａ１）などの集合管特異的な遺伝子の発現は確認されなかった。また、胎児における発生期の腎臓で発現するＰａｘ２やＰａｘ８などの遺伝子の発現が確認された。 As can be seen from Table 1, in urine falling cells, expression of proximal tubule-specific genes such as aquaporin 1, Na + / Cl − cotransporter (KCC3a), N-cadherin and the like was confirmed. Furthermore, Na + / HCO 3 expressed in proximal tubule and Henle's loop - cotransporter (SLC4A4), K- cadherin, Na + / HCO 3 expressed in Henle's loop and the distal tubule - cotransporter (SLC4A7), Gene expression such as calbindin D28K that is specifically expressed in the distal tubule was also confirmed. On the other hand, and renal corpuscle specific genes such as nephrin and P- cadherin, aquaporin 2 and Na + / HCO 3 - cotransporter (SLC4A1) Expression of collecting pipe specific gene, such as was not confirmed . In addition, expression of genes such as Pax2 and Pax8 expressed in the developing kidney of the fetus was confirmed.

これらの結果から、尿中落下細胞の初代培養細胞中には、発生期の腎臓に存在する腎臓幹／前駆細胞のような未分化細胞や、近位尿細管＞ヘンレループ＞遠位尿細管と続く腎ネフロンの尿細管細胞様に分化した細胞が存在することが示唆された。   From these results, the primary cultured cells of urinary falling cells are followed by undifferentiated cells such as kidney stem / progenitor cells present in the developing kidney, and proximal tubule> Henleloop> distal tubule It was suggested that cells differentiated like renal tubular cells of renal nephrons.

尿中落下細胞からの腎臓幹／前駆細胞の分離
ＤＮＡ結合色素であるヘキスト３３３４２（Hoechst33342）は、紫外線レーザー光で励起すると４００〜６００ｎｍ以上に亘る広範囲の蛍光を発することが知られており、多種の細胞が混ざったヘテロな細胞集団、例えば骨髄細胞等をこの色素で染色した後にフローサイトメトリーを用いて４５０ｎｍ前後の青色蛍光と６７５ｎｍ前後の赤色蛍光とで二次元に展開すると、通常の細胞周期解析で見られるＧ０／Ｇ１期の細胞よりもさらに蛍光の暗い部分に特異なパターンを持つヘキスト３３３４２弱陽性又は陰性の細胞集団が現れる。この細胞集団は、残りの大部分の細胞が属する主集団（メインポピュレーション)から突出した展開パターンを示すため、サイドポピュレーション（side population；SP）細胞と呼ばれる。 Hoechst 33342 (Hoechst33342), a DNA binding dye that separates kidney stem / progenitor cells from urinary falling cells, is known to emit a wide range of fluorescence over 400 to 600 nm when excited with ultraviolet laser light. When a heterogeneous cell population, such as bone marrow cells, is mixed with this dye and developed in two dimensions with blue fluorescence around 450 nm and red fluorescence around 675 nm using flow cytometry, the normal cell cycle A Hoechst 33342 weakly positive or negative cell population having a specific pattern in a darker fluorescence portion than the G0 / G1 phase cells observed in the analysis appears. This cell population is called a side population (SP) cell because it shows a development pattern that protrudes from the main population to which most of the remaining cells belong.

骨髄細胞においては、このＳＰ細胞中に造血幹細胞が濃縮されていることが知られている（文献「Goodell M. A. et al., J. Exp. Med., vol.183, p.1797, 1996」等を参照）。また、骨髄以外に、脳や骨格筋、心臓、肝臓、腎臓などの組織中でもＳＰ細胞が見つかっており（文献「Murayama A. et al., J. Neurosci. Res., vol.69, p.837, 2002」、文献「Gussoni E. et al., Nature, vol.401, p.390, 1999」、文献「Hierlihy A. M. et al., FEBS Lett., Vol.530, p.239, 2002」、文献「Asakura A. et al., Exp. Hematol., vol.30, p.1339, 2002」、文献「Iwatani H. et al., Kidney Int., vol.65, p.1604, 2004」等を参照）、これらの細胞でも組織幹細胞として機能している可能性がある。   In bone marrow cells, it is known that hematopoietic stem cells are concentrated in the SP cells (references such as “Goodell MA et al., J. Exp. Med., Vol. 183, p. 1797, 1996”). See). In addition to bone marrow, SP cells have also been found in tissues such as brain, skeletal muscle, heart, liver, and kidney (see “Murayama A. et al., J. Neurosci. Res., Vol.69, p.837). , 2002, literature `` Gussoni E. et al., Nature, vol.401, p.390, 1999 '', literature `` Hierlihy AM et al., FEBS Lett., Vol.530, p.239, 2002 '', literature See "Asakura A. et al., Exp. Hematol., Vol.30, p.1339, 2002", literature "Iwatani H. et al., Kidney Int., Vol.65, p.1604, 2004" etc. ), These cells may also function as tissue stem cells.

なお、ＳＰ細胞では、ＡＢＣトランスポーターであるＭＤＲ（multi drug resistance gene）がコードするタンパク質を代表とするポンプ状の分子によってヘキスト３３３４２が細胞外に排出される結果、ヘキスト３３３４２によって余り染色されないと考えられている。幹／前駆細胞は、ＭＤＲの発現が活発なため、ヘキスト３３３４２の排出能は幹／前駆細胞に共通の性質であることも示唆されている（実験医学、Vol.19, No.15（増刊）,p.68-73, 2001）。このＳＰ細胞画分は、ＭＤＲ分子の機能阻害剤であるベラパミルやレセルピンを添加すると完全に消失する（第１１７回日本医学会シンポジウム記録集、p.66-74）。   In SP cells, Hoechst 33342 is excreted to the outside by pump-like molecules typified by proteins encoded by ABC transporter MDR (multi drug resistance gene). It has been. Since stem / progenitor cells are actively expressing MDR, it is suggested that Hoechst 33342 excretion is a property common to stem / progenitor cells (Experimental Medicine, Vol.19, No.15 (extra number) , p.68-73, 2001). This SP cell fraction disappears completely when verapamil or reserpine, which is a function inhibitor of MDR molecules, is added (117th Annual Meeting of the Japanese Medical Society Symposium, p.66-74).

以上のような背景から、尿中落下細胞においても同様にＳＰ細胞が存在し、腎臓幹／前駆細胞として機能している可能性が考えられるため、尿中落下細胞からのＳＰ細胞の分離を試みた。   Against this background, there is a possibility that SP cells are also present in urinary falling cells and function as kidney stem / progenitor cells. Therefore, separation of SP cells from urinary falling cells was attempted. It was.

詳細には、上述のように初代培養を３週間続けた尿中落下細胞をトリプシン−ＥＤＴＡ溶液（Invitrogen）を用いて培養皿から剥がし、１×１０６個／ｍｌとなるように２％ ＦＢＳを含有するＤＭＥＭに懸濁したものを２本調製した。そして、各サンプルにヘキスト３３３４２（Morecular Probe）を５ｍｇ／ｍｌとなるように添加し、一方のサンプルのみにレセルピン（Sigma）を１ｍｇ／ｍｌとなるように添加して陰性コントロールとした。その後、細胞を３７℃で１時間、振盪培養した後、遠心分離して上清を除去し、上清除去後の細胞を、死細胞を選別する目的でＰＩ（propidium iodide）を１ｍｇ／ｍｌとなるように添加した、２％ ＦＢＳ、１０ｍＭ ＨＥＰＥＳ（Invitrogen）、１０２Ｕ／ｍｌ ペニシリン−ストレプトマイシン（Invitrogen）を含有するＨＢＳＳ（Hank's Balanced Salt Solution）で懸濁し、蛍光活性化セルソーター（BD FACSAria、Beckton Dickinson）を用いてＳＰ細胞の解析、分離を行った。 Specifically, as described above, fallen cells in urine that had been in primary culture for 3 weeks were detached from the culture dish using trypsin-EDTA solution (Invitrogen), and 2% FBS was added so that the concentration was 1 × 10 6 cells / ml. Two suspensions in DMEM were prepared. Then, Hoechst 33342 (Morecular Probe) was added to each sample so as to be 5 mg / ml, and reserpine (Sigma) was added to only one sample so as to be 1 mg / ml to serve as a negative control. Thereafter, the cells are cultured with shaking at 37 ° C. for 1 hour, and then centrifuged to remove the supernatant. After removing the supernatant, PI (propidium iodide) is added to 1 mg / ml for the purpose of selecting dead cells. Suspended with HBSS (Hank's Balanced Salt Solution) containing 2% FBS, 10 mM HEPES (Invitrogen), 10 2 U / ml penicillin-streptomycin (Invitrogen), and added to a fluorescence activated cell sorter (BD FACSAria, Beckton SP cells were analyzed and separated using Dickinson).

ＳＰ細胞の解析結果を図３に示す。図３（Ａ）はレセルピンを添加していないサンプルを示し、図３（Ｂ）はレセルピンを添加した陰性コントロールのサンプルを示す。図中、実線で囲んだ領域がヘキスト３３３４２弱陽性又は陰性画分である。この画分は、レセルピンを添加した陰性コントロールでは完全に消失しているため、この画分がＳＰ細胞画分であることが確認された。なお、尿中落下細胞中には平均で０．３３％（ｎ＝６）のＳＰ細胞が存在した。   The analysis result of SP cells is shown in FIG. FIG. 3A shows a sample to which no reserpine was added, and FIG. 3B shows a negative control sample to which reserpine was added. In the figure, the region surrounded by the solid line is Hoechst 33342 weak positive or negative fraction. Since this fraction disappeared completely in the negative control to which reserpine was added, it was confirmed that this fraction was the SP cell fraction. In addition, 0.33% (n = 6) SP cells were present on average in the urine falling cells.

このＳＰ細胞を分離・取得した後、初代培養時と同じ方法で培養を行った。細胞を経時的に顕微鏡観察した結果を図４に示す。図４（Ａ）〜（Ｆ）は、それぞれ１２時間、３日、１０日、１５日、１９日、２２日経過後の細胞を示したものである。培養を行うと、高増殖能を持ち、且つ近位尿細管上皮細胞様の敷石状形態を有する上皮性細胞コロニーが得られ、さらに培養を続けたところ、２２日経過後には細胞ドームの形成が確認された。一方、ＳＰ細胞画分以外の細胞を同様の方法で培養しても、細胞ドームの形成は確認されなかった。   After the SP cells were isolated and obtained, the cells were cultured in the same manner as in the primary culture. The results of microscopic observation of the cells over time are shown in FIG. 4A to 4F show cells after 12 hours, 3 days, 10 days, 15 days, 19 days, and 22 days, respectively. When cultured, an epithelial cell colony having a high proliferative capacity and a proximal tubule epithelial cell-like paving stone-like morphology was obtained, and further culturing revealed that cell dome formation occurred after 22 days. confirmed. On the other hand, even when cells other than the SP cell fraction were cultured by the same method, formation of a cell dome was not confirmed.

これらの結果から、初代培養した尿中落下細胞からヘキスト３３３４２弱陽性又は陰性のＳＰ細胞を分離することにより、尿細管上皮細胞に分化し得る腎臓幹／前駆細胞を分離・取得できることが示された。   From these results, it was shown that kidney stem / progenitor cells that can differentiate into tubular epithelial cells can be separated and obtained by isolating Hoechst 33342 weakly positive or negative SP cells from primary cultured urine falling cells. .

尿中落下細胞からの腎臓幹／前駆細胞分離の効率化
次に、尿中落下細胞からの腎臓幹／前駆細胞の分離効率化を検討するため、近年、腎疾患の指標となり得ることが報告された尿中Ｌ−ＦＡＢＰ値と、初代培養の培養効率との関係を検討した。 Efficient separation of kidney stem / progenitor cells from urinary falling cells Next, in order to investigate the efficiency of separation of kidney stem / progenitor cells from urinary falling cells, it has recently been reported that it can be an indicator of kidney disease. The relationship between the urinary L-FABP value and the culture efficiency of the primary culture was examined.

詳細には、腎機能の未熟な新生児を含む２３９例の患者由来の新鮮尿から分離された尿中落下細胞を上述と同様に初代培養し、その培養効率を尿中Ｌ−ＦＡＢＰ値と比較した。培養１週間の時点で上皮様細胞のコロニー形成を認め、且つ３週間まで増殖の見られたものを初代培養成功例とした。尿中Ｌ−ＦＡＢＰ値によりその培養効率を比較したところ、１群：Ｌ−ＦＡＢＰ＜１００ｎｇ／ｍｌで１５％、２群：１００＜Ｌ−ＦＡＢＰ＜１０００ｎｇ／ｍｌで２２％、３群：１０００＜Ｌ−ＦＡＢＰ＜１００００ｎｇ／ｍｌで３５％、４群：Ｌ−ＦＡＢＰ＞１００００ｎｇ／ｍｌで１００％となった。   Specifically, urine falling cells isolated from fresh urine derived from 239 patients including neonates with immature kidney function were cultured in the same manner as described above, and the culture efficiency was compared with urinary L-FABP levels. . A case where epithelial cell colony formation was observed at 1 week of culture and growth was observed up to 3 weeks was regarded as a successful primary culture. When the culture efficiency was compared by the urinary L-FABP value, the 1st group: 15% when L-FABP <100 ng / ml, the 2nd group: 22% when 100 <L-FABP <1000 ng / ml, and the 3rd group: 1000 < L-FABP <10000 ng / ml was 35%, Group 4: L-FABP> 10000 ng / ml was 100%.

初代培養細胞は、アルカリホスファターゼ染色陽性を示し、経上皮輸送能を示す細胞ドームの形成が確認された。また、初代培養細胞からｍＲＮＡを抽出し、ＲＴ−ＰＣＲ法により遺伝子発現を確認したところ、近位尿細管特異的アミノトランスポーターｒＢＡＴ（related to b0,+ system amino acid transporter）の発現が確認された。   The primary cultured cells were positive for alkaline phosphatase staining, confirming the formation of a cell dome exhibiting transepithelial transport ability. In addition, when mRNA was extracted from primary cultured cells and gene expression was confirmed by RT-PCR, the expression of the proximal tubule-specific amino transporter rBAT (related to b0, + system amino acid transporter) was confirmed. .

これらの結果から、尿中Ｌ−ＦＡＢＰを指標にすることにより、その高値尿から効率的に尿細管細胞に分化し得る尿中落下細胞の初代培養が可能になることが示された。   From these results, it was shown that by using urinary L-FABP as an index, primary culture of urinary falling cells that can be efficiently differentiated from high-value urine into tubular cells becomes possible.

さらに、尿中Ｌ−ＦＡＢＰ値と、ＳＰ細胞の分離効率との関係についても検討した。この結果、ＳＰ細胞の割合は、図５に示すように、初代培養時の尿中Ｌ−ＦＡＢＰ値と非常に高い相関（Ｒ＝０．８２）を示すことが明らかにされた。   Furthermore, the relationship between the urinary L-FABP value and the SP cell separation efficiency was also examined. As a result, it was clarified that the proportion of SP cells showed a very high correlation (R = 0.82) with the urinary L-FABP value during primary culture, as shown in FIG.

このことは、腎疾患患者において尿中Ｌ−ＦＡＢＰ値が上昇している時期を特定し採尿することにより、腎臓幹／前駆細胞が取得できる可能性を示唆しており、非侵襲的且つ効率的なヒト腎細胞の樹立に大きく貢献する方法が見出されたものと考えられる。   This suggests the possibility that kidney stem / progenitor cells can be obtained by identifying the time when urinary L-FABP levels are rising in patients with renal disease and collecting urine, which is noninvasive and efficient. It is considered that a method that greatly contributes to the establishment of new human kidney cells has been found.

腎臓幹／前駆細胞の腎臓への生着
次に、分離した腎臓幹／前駆細胞が成体腎臓に生着し、機能する形質を有しているか否かを確認するため、成体腎臓に障害を与え、腎臓が再生する過程においてこの細胞が生着するか否かを検討した。 Engraftment to the kidneys of renal stem / progenitor cells Next, since the renal stem / progenitor cells isolated are engrafted in adult kidney, confirms whether a trait that functions, harm to the adult kidney In the process of regenerating the kidney, it was examined whether or not the cells were engrafted.

詳細には、ヒト腎臓幹／前駆細胞を異種移植するレシピエントとして、拒絶反応を起しにくい実験的免疫不全マウス（２５ｇ）を使用し、両腎臓の腎動静脈を駆血し、３０分後虚血を解除し、虚血性急性腎不全モデルを作製した。分離したＳＰ細胞をさらに５週間培養し、トリプシン−ＥＤＴＡ溶液（Invitrogen）を用いて単一細胞にまで分散させた後、虚血解除した直後の腎皮膜下経路により注射器にて細胞移植を行った。そして、３日後に剖検して腎臓を摘出し、凍結切片を作成した。   Specifically, as a recipient for xenotransplantation of human kidney stem / progenitor cells, experimental immunodeficient mice (25 g) that are difficult to cause rejection are used, and the renal arteries and veins of both kidneys are fed, and 30 minutes later. Ischemia was released and an ischemic acute renal failure model was created. The separated SP cells were further cultured for 5 weeks, dispersed into single cells using a trypsin-EDTA solution (Invitrogen), and then transplanted with a syringe through the subrenal capsule route immediately after the release of ischemia. . Three days later, necropsy was performed to remove the kidney, and a frozen section was prepared.

ヒト特異的ＨＬＡ抗体（緑色蛍光標識）による免疫染色と、尿細管特異的アクアポリン１抗体（赤色蛍光標識）による免疫染色との２重染色を行った結果を図６に示す。図中、黄色を呈した部位は、移植細胞が尿細管様の管腔構造を形成した共陽性の部位である。虚血性急性腎不全モデルに異種移植を実施したにも拘わらず、生命予後の悪化は観察されなかった。   FIG. 6 shows the result of double staining of immunostaining with a human-specific HLA antibody (green fluorescent label) and immunostaining with a tubule-specific aquaporin 1 antibody (red fluorescent label). In the figure, the yellow-colored site is a co-positive site where the transplanted cells formed a tubular-like lumen structure. Despite xenotransplantation in an ischemic acute renal failure model, no worsening of life prognosis was observed.

これらの結果から、分離した腎臓幹／前駆細胞は成体腎臓に生着し、尿細管特異的な機能を有するタンパクを発現する形質を有していることが示された。したがって、この腎臓幹／前駆細胞を含む腎疾患治療剤を腎疾患患者に移植することで、腎疾患を治療することができると考えられる。   From these results, it was shown that the isolated kidney stem / progenitor cells were engrafted in the adult kidney and had a trait expressing a protein having a tubule-specific function. Therefore, it is considered that renal disease can be treated by transplanting a renal disease therapeutic agent containing kidney stem / progenitor cells to a renal disease patient.

以上、具体的な実験結果を参照しながら説明したように、本実施の形態の方法によれば、腎疾患患者から非侵襲的に効率よく腎臓幹／前駆細胞を分離し、分離した腎臓幹／前駆細胞を提供することができる。また、その腎臓幹／前駆細胞を含む腎疾患治療剤を提供することができる。特に、本実施の形態の方法では、腎疾患患者自身から腎臓幹／前駆細胞を分離することができるため、分離した腎臓幹／前駆細胞をその患者に移植することもでき、腎疾患に対するオーダーメイド医療の開発に応用することができると考えられる。さらに、分離した腎臓幹／前駆細胞を培養して得られたヒト尿細管細胞培養系は、in vitroでの薬剤スクリーニングにも使用することができる。   As described above with reference to the specific experimental results, according to the method of the present embodiment, the kidney stem / progenitor cells are efficiently and non-invasively separated from the kidney disease patient, and the isolated kidney stem / Progenitor cells can be provided. In addition, a therapeutic agent for renal diseases comprising the kidney stem / progenitor cells can be provided. In particular, in the method of the present embodiment, the kidney stem / progenitor cells can be isolated from the kidney disease patient itself, so that the isolated kidney stem / progenitor cells can also be transplanted into the patient, making it custom-made for kidney disease. It can be applied to medical development. Furthermore, the human tubular cell culture system obtained by culturing isolated kidney stem / progenitor cells can be used for in vitro drug screening.

なお、本発明は上述した実施の形態のみに限定されるものではなく、本発明の要旨を逸脱しない範囲において種々の変更が可能であることは勿論である。   It should be noted that the present invention is not limited to the above-described embodiments, and various modifications can be made without departing from the scope of the present invention.

腎疾患患者から分離した尿中落下細胞を培養し、経時的に顕微鏡観察した結果を示す図である。It is a figure which shows the result of having culture | cultivated the fall cell in the urine isolate | separated from the patient with a renal disease, and observing with a microscope over time. 尿中落下細胞を培養することにより形成される細胞ドームを示す図である。It is a figure which shows the cell dome formed by culturing the fall cell in urine. 尿中落下細胞をヘキスト３３３４２で染色したときの弱陽性又は陰性画分を示す図である。It is a figure which shows a weak positive or negative fraction when the fallen cell in urine is dye | stained with Hoechst 33342. 分離したヘキスト３３３４２弱陽性又は陰性のＳＰ細胞を培養し、経時的に顕微鏡観察した結果を示す図である。It is a figure which shows the result of having culture | cultivated the separated Hoechst 33342 weakly positive or negative SP cell, and observing it microscopically. 尿中Ｌ−ＦＡＢＰ値とＳＰ細胞の分離割合との関係を示す図である。It is a figure which shows the relationship between the urinary L-FABP value and the isolation | separation ratio of SP cell. 分離したＳＰ細胞の腎臓への生着を示す図である。It is a figure which shows the engraftment to the kidney of the isolate | separated SP cell.

Claims (3)

尿中の肝型脂肪酸結合タンパク質が高値を示すヒト腎疾患患者の尿中に含まれる細胞の初代培養細胞をヘキスト３３３４２で染色し、その弱陽性又は陰性画分を分離することを特徴とする腎臓幹／前駆細胞の分離方法。 A kidney characterized in that primary cultured cells of cells contained in the urine of a human kidney disease patient having a high level of liver-type fatty acid binding protein in urine are stained with Hoechst 33342 and the weakly positive or negative fraction is separated. Stem / progenitor cell separation method. 上記弱陽性又は陰性画分は、上記ヘキスト３３３４２と共にＭＤＲ（ｍｕｌｔｉ ｄｒｕｇ ｒｅｓｉｓｔａｎｃｅ ｇｅｎｅ）分子の機能阻害剤を添加することにより消失する画分であることを特徴とする請求項１記載の腎臓幹／前駆細胞の分離方法。   The kidney stem / precursor according to claim 1, wherein the weakly positive or negative fraction is a fraction that disappears by adding a function inhibitor of MDR (multi drug resistance gene) molecule together with Hoechst 33342. Cell separation method. 初代培養の培地には、マウス間葉系細胞（受託番号ＦＥＲＭ ＢＰ−１０８６５）の培養上清が含まれていることを特徴とする請求項１記載の腎臓幹／前駆細胞の分離方法。   The method for separating kidney stem / progenitor cells according to claim 1, wherein the primary culture medium contains a culture supernatant of mouse mesenchymal cells (Accession No. FERM BP-10865).
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